Herpes simplex virus (HSV) is a common human pathogen causing lifelong infection. HSV infects and replicates in epithelial cells before spreading to sensory neurons where it establishes a lifelong latency punctuated by frequent reactivation events. Cytotoxic cells of the innate and adaptive immune systems play critical roles in limiting primary and recurrent viral spread and replication in epithelia. Furthermore, cytotoxic CD8+ T cells control the extent of viral reactivation from latently infected neurons. My goal is t learn how HSV affects the function of cytotoxic cells by altering key immunoregulatory functions of its two main entry receptors: HVEM and nectin-1. It is remarkable that both receptors are also involved in complex networks of interactions that balance the activation and inhibition of cytolyti cells. HVEM is at the crossroad between TNFR and CD28 co-modulatory networks of T cells during antigen recognition. Nectin-1 is a ligand for the NK cell activating receptor CD96 but its role in innate immunity remains relatively unclear. I showed that binding of the viral ligand, HSV gD, can affect the function of HVEM and nectin-1 in two ways: by competing with natural ligands and by inducing receptor endocytosis. Thus, my general hypothesis is that HSV uses gD as a non-canonical ligand to alter the host's defenses. In this grant I will compare and contrast how gD affects the function of CTL and NK cells towards infected cells. The proposed aims will focus on molecular interactions (aim 1), target recognition (aim 2), and functional activation (aim 3). Aim 1 is based on my preliminary data that compare the structures of HVEM and nectin-1 bound to gD or to their respective natural ligands. The gD binding sites overlap but differ from the functional sites of the receptors suggesting that gD can compete with natural ligands and induce a specific response. In Aim 2, I will study how gD perturbs the contacts between cytolytic cells and their targets by affecting nectin-1 and HVEM at the NK and CTL immunological synapses respectively. To address NK and T cell function in Aim 3, I established collaborations with Drs J. Orange and J. Riley (both at PENN) and Dr D. Nardelli Haefliger (U. of Lausanne). I will investigate how gD affects the activation of NK cells and modulates CD8 T cell cytotoxicity in the presence or absence of antigen presentation. Successful completion of this study will define how gD, a key entry glycoprotein functions in two novel immune evasion strategies for HSV by targeting key regulators of the innate and adaptive immune responses. I have proposed testable hypotheses to understand the immunological role of gD during HSV infection and to use it as a tool to unravel the complex immunoregulatory networks involved in cytolytic activity of NK and T cells in general. This molecular immunology approach will open possibilities to address these functions in vivo. Furthermore, data from this study may suggest ways to design novel antiviral interventions.